A Shared Mechanism for the Folding of Voltage-Gated K+ Channels

Abstract

In this study, we probe the folding of K_vAP, a voltage-gated K⁺ (K_v) channel. The K_vAP channel, though of archaebacterial origin, is structurally and functionally similar to eukaryotic K_v channels. An advantage of the K_vAP channel is that it can be folded in vitro from an extensively unfolded state and the folding can be controlled by temperature. We utilize these properties of the K_vAP channel to separately study the membrane insertion and the tetramerization stages during folding. We use two quantitative assays: a Cys PEGylation assay to monitor membrane insertion and a cross-linking assay to monitor tetramerization. We show that during folding the K_vAP polypeptide is rapidly inserted into the lipid bilayer with a "native-like" topology. We identify a segment at the C-terminus that is important for multimerization of the K_vAP channel. We show that this C-terminal domain forms a dimer, which raises the possibility that the tetramerization of the K_vAP channel proceeds through a dimer of dimers pathway. Our studies show that the in vitro folding of the K_vAP channel mirrors aspects of the cellular assembly pathway for voltage-gated K⁺ channels and therefore suggest that evolutionarily distinct K_v channels share a common folding pathway. The pathway for the folding and assembly of a K_v channel is of central importance as defects in this pathway have been implicated in the etiology of several disease states. Our studies indicate that the K_vAP channel provides an experimentally tractable system for elucidating the folding mechanism of K_v channels.

Figure 1.. Structure and folding of the K_vAP channel.

(A) Top view of the tetrameric K_vAP channel. The structural model of the K_vAP channel previously described is shown. The structure illustrates the domain swap between the pore and voltage sensor domains of adjacent subunits in the K_vAP channel. (B) Topology map illustrating the domains and the structural features of a K_vAP channel subunit. Dashed lines are membrane boundaries. (C) The folding pathway of the K_vAP channel involves the stages of membrane insertion, secondary/tertiary folding and tetramerization. Structure figures were generated with VMD.

Figure 2.. In vitro folding of the K_vAP channel is dependent on temperature.

(A, B) Time course of the in vitro folding of the K_vAP channel at 20 °C (A) and at 80 °C (B). Representative SDS-PAGE gels showing glutaraldehyde crosslinking of the in vitro folding reaction at various time points over 2 hours is shown. The last lane is a time point at 2 hours without glutaraldehyde added (−). The oligomeric nature of the crosslinked band is indicated. (C) Tetramerization kinetics of the K_vAP channel at different temperatures are plotted. Data have been fitted with either a single (40 °C and 80 °C) or a double exponential (50 °C). Data at 20 °C could not be fitted to an exponential. (D) Temperature downshift arrests folding of K_vAP. The in vitro folding reaction for the K_vAP channel was carried out at 50 °C. After incubation for 2, 5 or 16 minutes, the samples were transferred from 50 °C to 20 °C and the tetramerization kinetics were followed. No additional tetramerization was observed at 20 °C. The data points following the downshift are fitted with a straight line. Error bars shown in C and D are standard deviations for n =3 using at least 2 independent protein preparations.

Figure 3.. The Cys-PEGylation assay used to assess the membrane insertion of K_vAP.

(A) The reaction scheme used in Cys-PEGylation assay is shown. (B) Topology map of the VSD of the K_vAP channel illustrating the cysteines used to demonstrate the methodology. 37C is transmembrane and 143C is in a loop outside of the membrane. (C) SDS-PAGE gels showing the results of the Cys-PEGylation for 37C and 143C. (D) PEGylation results for 37C and D143C were quantified and plotted as bar graphs. Error bars are standard deviations from n=3 experiments using at least 2 independent protein preparations.

Figure 4.. Membrane insertion of K_vAP during folding.

(A) Topology map of the K_vAP channel showing the location of the Cys substitutions used for the PEGylation assays. (B) Bar graph showing the PEGylation results for the Cys substitutions. Sites colored in gold are transmembrane, blue are in loops outside of the membrane and red are sites in the pore helix. Error bars are standard deviations from n=3 experiments using at least 2 independent protein preparations. Some error bars are zero because the measured reactivity was zero.

Figure 5.. Deletion of the CTD abolishes tetramerization of K_vAP in POPC lipids.

(A) The C-terminal sequences of the K_vAP channel constructs used are shown. All constructs contain the wild type K_vAP channel residues 1 – 238. The S6 transmembrane segment in the K_vAP channel ends at residue 237. The Lys residues introduced to enhance glutaraldehyde crosslinking are indicated in red. (B) Time course of in vitro folding of the K_vAPΔCTD+ Lys channel and the full length control in POPC lipid vesicles at 50 °C. Shown to the right are schematic illustrations of the constructs used. Data for the full-length K_vAP channel is from Figure 2. Tetramerization kinetics were quantitated from n=3 experiments with at least 2 protein preparations. Error bars are standard deviations. Tetramerization kinetics for K_vAPΔCTD+Lys could not be fitted with an exponential equation.

Figure 6.. The CTD of K_vAP is a dimer.

(A) Schematic illustration of the SUMO-GCN4 and SUMO-K_vAP CTD constructs used. (B) SEC of the SUMO-K_vAP-CTD, SUMO-GCN4 and SUMO proteins show that the SUMO- K_vAP-CTD is a dimer, SUMO-GCN4 is a tetramer while the SUMO protein migrates as a monomer. (C) SDS-PAGE gel showing the glutaraldehyde cross-linking of the SUMO- K_vAP-CTD and SUMO-GCN4 proteins confirms that the SUMO- K_vAP-CTD is a dimer while SUMO-GCN4 is a tetramer.

Figure 7.. The CTD inhibits in vitro folding of K_vAP.

(A) The reaction scheme used in the competition assay is shown. The competitors used are the S6-CTD or the S6 peptide. The schematic representation of the proteins used in the assay are shown. (B) SDS-PAGE gel showing the glutaraldehyde crosslinking of the K_vAP folding reaction carried out in the presence of the S6+CTD or the S6 peptide. The identity of the protein bands observed on glutaraldehyde crosslinking are indicated. The protein bands indicated by the asterisk potentially corresponds to a K_vAP+S6-CTD dimer and a S6-CTD tetramer based on their molecular weights. Control lanes where the competitor was added after completion of folding are marked with a C. A five-fold molar ratio of competitor to unfolded K_vAP is used in all lanes. Monomers of S6-CTD and the S6 peptide are not visible on this gel because of their low molecular weight. A decrease in the density of the K_vAP tetramer band is seen in the case of folding in the presence of S6-CTD but not in the presence of the S6 peptide. (C) Inhibition of the in vitro folding reaction. Bar graph showing the % of tetramer inhibition observed in the presence of the S6+CTD and the S6 peptide. Error bars correspond to the standard deviation for n = 3 from at least 2 protein preparations.

Figure 8:. Dimerization of the CTD is not dependent on temperature.

(A) The reaction scheme used for the CTD dimerization assay. The SUMO-CTD construct is used in this assay for easy visualization on an SDS-PAGE gel. (B) SDS-PAGE gel showing the extent of dimerization of SUMO-CTD as determined by glutaraldehyde crosslinking. The dimeric SUMO-CTD is dissociated by treatment with 0.35 % SDS, re-associated at the indicated temperature and the extent of re-association is determined by glutaraldehyde crosslinking. Glutaraldehyde crosslinking of the native SUMO-CTD before (−) and after treatment (+) with 0.35% SDS is also shown. Glutaraldehyde crosslinking of the monomeric and the dimeric SUMO-CTD proteins results in an additional band that is indicated by an asterisk. (C) The extent of SUMO-CTD dimerization at the different temperatures were quantified and plotted as a bar graph. The error bars are standard deviations from three experiments with two different protein preparations.